Pathological and mammographic prognostic factors for screen detected cancers in a multi-centre randomised, controlled trial of mammographic screening in women from age 40 to 48 years.

AIM: To assess pathological and radiological prognostic factors for cancers detected by screening within a multi-centre RCT trial of mammographic screening of younger women. METHOD: The survival of 232 women with screen detected invasive cancer was ascertained. Data on invasive cancer size, histological grade, nodal status, vascular invasion, mammographic spiculation, comedo calcification and mammographic background were assessed. Kaplan-Meier and Cox proportional hazards methods were used to examine survival. RESULTS: Univariate analysis indicated that women with cancers with the following features had poorer survival; ≥ 30 mm, histologically grade 3, heavily node positive (4 or more positive nodes), vascular invasion positive and displaying mammographic comedo calcification. In a multivariate model survival remained poorer in women with four or more nodes positive (HR 8.36, 95% CI 2.31, 30.17) and in those with comedo calcification (HR 3.00,95% CI 1.13, 7.99). CONCLUSION: Nodal status and the presence of mammographic comedo calcification have independent prognostic significance in young women with screen detected cancer.

The value of immunohistochemistry in sentinel lymph node histopathology in breast cancer.

The optimal protocol for the histopathological examination of sentinel lymph nodes (SLNs) in breast cancer has not been determined. The value of more detailed examination using immunohistochemistry (IHC) is controversial. A total of 476 SLNs from 216 patients were reviewed. Sentinel lymph nodes were sectioned at three levels at 100 mum intervals and stained with haematoxylin and eosin (H&E). If the H&E sections showed no evidence of metastasis, then the three serial sections were stained with a murine monoclonal anti-cytokeratin antibody (CAM 5.2). Metastatic deposits were classified as macrometastasis (> 2.0 mm), micrometastasis (0.2-2.0 mm) or isolated tumour cells (ITC, < 0.2 mm). Of the 216 patients, 56 (26%) had metastasis as identified by H&E. Immunohistochemistry detected metastatic deposits in a further nine patients (4%), of whom four (2%) had micrometastasis and five (2%) had ITC only. Those cases with micrometastases were all, on review, visible on the H&E sections. Immunohistochemistry detects only a small proportion of metastasis in SLNs. All metastatic deposits identified by IHC were either micrometastasis or ITC. Until the prognostic significance of these deposits has been determined, IHC may be of limited value in the histopathological examination of SLNs.

Can breast MRI help in the management of women with breast cancer treated by neoadjuvant chemotherapy?

Contrast-enhanced (CE) MRI was used to monitor breast cancer response to neoadjuvant chemotherapy. Patients underwent CE MRI before and after therapy, together with conventional assessment methods (CAM). CE MRI was carried out at 1.5 T in the coronal plain with 3D sequences before and after bolus injection. An expert panel determined chemotherapy response using both CE MRI and CAM. Histopathological response in the surgical specimen was then used to determine the sensitivity and specificity of CE MRI and CAM. In total, 67 patients with 69 breast cancers were studied (mean age of 46 years). Tumour characteristics showed a high-risk tumour population: median size 49 mm: histopathological grade 3 (55%): oestrogen receptor (ER) negative (48%). Histopathological response was as follows: - complete pathological response (pCR) 17%; partial response (pPR) 68%; no response (NR) 15%. Sensitivity of CAM for pCR or pPR was 98% (CI 91-100%) and specificity was 50% (CI 19-81%). CE MRI sensitivity was 100% (CI 94-100%), and specificity was 80% (CI 44-97%). The absolute agreement between assessment methods and histopathology was marginally higher for CE MRI than CAM (81 vs 68%; P=0.09). In 71%, CE MRI increased diagnostic knowledge, although in 20% it was judged confusing or incorrect. The 2nd MRI study significantly increased diagnostic confidence, and in 19% could have changed the treatment plan. CE MRI persistently underestimated minimal residual disease. In conclusion, CE MRI of breast cancer proved more reliable for predicting histopathological response to neoadjuvant chemotherapy than conventional assessment methods.

Degenerate oligonucleotide primed-polymerase chain reaction-based array comparative genomic hybridization for extensive amplicon profiling of breast cancers : a new approach for the molecular analysis of paraffin-embedded cancer tissue.

We have developed a protocol for degenerate oligonucleotide-primed-polymerase chain reaction-based array comparative genomic hybridization (array CGH) that, when combined with a laser microdissection technique, allows the analysis of cancer cell populations isolated from routine, formalin-fixed, paraffin-embedded tissue samples. Comparison of copy number changes detected by degenerate oligonucleotide-primed-polymerase chain reaction-based array CGH to those detected by conventional array CGH or fluorescence in situ hybridization, demonstrated that amplifications can be reliably detected. Using a genomic microarray containing 57 oncogenes, we screened a total of 28 breast cancer samples and obtained a detailed amplicon profile that is the most comprehensive to date in human breast cancer. The array CGH method described here will allow the genetic analysis of paraffin-embedded human cancer materials for example in the context of clinical trials.

Study of interlaboratory reliability and reproducibility of estrogen and progesterone receptor assays in Europe. Documentation of poor reliability and identification of insufficient microwave antigen retrieval time as a major contributory element of unreliable assays.

Immunohistochemical assays for estrogen receptors (ERs) and progesterone receptors (PRs) have not been surveyed for technical validity. In the present study, the reliability of the immunohistochemical assay for ER and PR was evaluated using data from 105 laboratories participating in external quality assessment (EQA) during a 2-year period. Technical variables associated with reliable immunostaining were analyzed. The efficiency of the antigen retrieval step was identified as the single most important contributory factor influencing the overall reproducibility of the assays. Reliable assays were found in 24 (36%) of 66 laboratories participating in continual EQA, including the majority of centers known to have clinically validated results. Inadequate assay sensitivity, with subsequent weak staining, was the main cause of poor and variable results by laboratories using microwave antigen retrieval; too short a heating time was identified as the principal contributory factor. Extension of the heating time resulted in significant improvement regardless of all other variables in the immunohistochemical protocol. Continual participation in EQA is an effective means for identifying and ameliorating variables that influence the reliability of immunohistochemical assays for predictive markers, thereby assisting in technical validation and standardization.

Reliability of immunohistochemical demonstration of oestrogen receptors in routine practice: interlaboratory variance in the sensitivity of detection and evaluation of scoring systems.

AIMS: To investigate interlaboratory variance in the immunohistochemical (IHC) detection of oestrogen receptors so as to determine the rate of false negatives, which could adversely influence the decision to give adjuvant tamoxifen treatment. METHODS: To ensure that similar results are obtained by different institutions, 200 laboratories from 26 countries have joined the UK national external quality assessment scheme for immunocytochemistry (NEQAS-ICC). Histological sections from breast cancers having low, medium, and high levels of oestrogen receptor expression were sent to each of the laboratories for immunohistochemical staining. The results obtained were evaluated for the sensitivity of detection, first by estimating threshold values of 1% and 10% of stained tumour cells, and second by the Quick score method, by a panel of four assessors judging individual sections independently on a single blind basis. The results were also evaluated using participants' own threshold values. RESULTS: Over 80% of laboratories were able to demonstrate oestrogen receptor positivity on the medium and high expressing tumours, but only 37% of laboratories scored adequately on the low expressing tumour. Approximately one third of laboratories failed to register any positive staining in this tumour, while one third showed only minimal positivity. CONCLUSIONS: There is considerable interlaboratory variability, especially in relation to the detection of breast cancers with low oestrogen receptor positivity, with a false negative rate of between 30% and 60%. This variability appears to be caused by minor differences in methodology that may be rectified by fine adjustment of overall technique.

Minimal breast cancer: evaluation of histology and biological marker expression.

Ninety-eight minimal breast cancers (MBCs) diagnosed between 1975 and 1990, and all originally considered to be invasive were found, on review, to form three groups: (a) 28 predominantly invasive carcinomas < or = 10 mm ('predominant invasive'); (b) 48 predominantly ductal carcinoma in situ (DCIS) lesions with definite foci of invasion each < or = 10 mm ('predominant DCIS'); and (c) 22 DCIS without evidence of invasion ('pure DCIS'). Tumour histology and immunohistochemical expression of Ki-67, c-erbB2, p53, oestrogen receptor (ER), progesterone receptor (PR), and Bcl-2 were compared. The major finding was the contrasting features in the two invasive groups, with significant differences in their extent of invasion (P < 0.0001), tumour grade (P = 0.03), DCIS type (P = 0.008) and in marker expression. In the predominant invasive group, the infiltrative component was usually greater than 5 mm, low-grade and associated with well-differentiated DCIS. Expression of Ki-67, c-erbB2 and p53 was generally low, and that of ER, PR and Bcl-2 high. The predominant DCIS group in contrast had a much smaller, commonly high-grade, invasive component, usually with poorly differentiated DCIS and the reverse pattern of marker expression. Although not significant, survival of patients in the predominant invasive group was slightly better. These findings suggest that invasive MBCs should perhaps be treated as separate entities, in order to aid more appropriate selection of treatment.

Angiogenesis and expression of thymidine phosphorylase by inflammatory and carcinoma cells in ductal carcinoma in situ of the breast.

Angiogenesis is essential for tumour growth and important in metastasis and for prognosis in invasive carcinoma of the breast. Two patterns of increased vascularity have been shown in mammary ductal carcinoma in situ (DCIS): a cuff of vessels close to the involved ducts, and vessels in the interductal stroma. Inflammation may potentially promote angiogenesis by release of angiogenic factors and digestive enzymes. A correlation has previously been found between the intensity of perivascular inflammation and stromal vascularity in DCIS, but no strong relationship has been observed between inflammation and angiogenesis in invasive carcinoma. Tumour angiogenesis is regulated by a number of angiogenic factors, including thymidine phosphorylase (platelet-derived endothelial cell growth factor), which is expressed at high levels in macrophages. Using immunohistochemical methods, thymidine phosphorylase expression and vascularity have been studied in DCIS (n = 34) and invasive carcinoma (n = 32). Stromal vascularity in DCIS was associated with thymidine phosphorylase expression in the perivascular inflammatory cells and in the cytoplasm of carcinoma cells. In invasive carcinoma, no relationship was found between vascularity and thymidine phosphorylase expression in either the carcinoma or the inflammatory cells. This study suggests that thymidine phosphorylase expression in both inflammatory and carcinoma cells may contribute to one of the patterns of vascularity in DCIS, but not in invasive disease.

Increased frequency of TP53 mutations in BRCA1 and BRCA2 ovarian tumours.

We screened 81 ovarian tumours (30 BRCA1 associated, 18 BRCA2 associated, and 33 sporadic) for somatic TP53 mutations using both DNA analysis and immunostaining. TP53 mutations were significantly more frequent in tumours with mutations in BRCA1 (70% by immunostaining and 60% by DNA analysis) and BRCA2 (67% and 50%) compared to sporadic controls (39% and 30%) (P = 0.009). A higher proportion of tumours with BRCA1 and BRCA2 mutations were poorly differentiated, and TP53 mutant tumours in all categories were also more likely to be poorly differentiated. The poor differentiation of tumours with BRCA1 and BRCA2 mutations may be directly related to the role of these genes in DNA repair, and the need to overcome cell cycle checkpoints, often through loss of TP53. These results are consistent with the model of BRCA-induced tumorigenesis in which loss of checkpoint control is necessary for tumour development.

Invasive lobular and invasive ductal carcinoma of the breast show distinct patterns of vascular endothelial growth factor expression and angiogenesis.

Angiogenesis is essential for tumour growth and important in tumour metastasis and prognosis. Vascular endothelial growth factor (VEGF) stimulates endothelial proliferation in vitro and angiogenesis in vivo. VEGF expression has been correlated with high vascularity in tumours, including carcinoma of the breast. This study investigated VEGF expression and vascularity of invasive lobular (n = 10) and invasive ductal carcinoma (n = 28), and pure ductal carcinoma in situ of the breast (n = 33). VEGF protein expression was studied with immunohistochemistry and VEGF mRNA with in situ hybridization. Vascular density was assessed on sections stained for von Willebrand factor. There was more expression of both VEGF protein (P = 0.006) and mRNA (P = 0.002) in invasive ductal than in invasive lobular carcinoma. VEGF protein (rs = 0.32, P = 0.047) and mRNA (rs = 0.56, P = 0.04) correlated with vascular density in invasive ductal carcinoma. In invasive lobular carcinoma, vascular density did not correlate with VEGF mRNA (rs = 0.15, P = 0.35) and was inversely related to VEGF protein (rs = -0.57, P = 0.04). There were no significant differences in vascular density between the two types of invasive carcinoma, suggesting that VEGF is important in angiogenesis in invasive ductal carcinoma, but that other angiogenic factors are important in invasive lobular carcinoma. Although VEGF protein was frequently expressed in ductal carcinoma in situ, no relationship was found between VEGF and the two patterns of angiogenesis previously described.

Angiogenesis and inflammation in invasive carcinoma of the breast.

AIM: To investigate the relation between angiogenesis and inflammation in invasive carcinoma of the breast. METHODS: Sections from 75 invasive carcinomas of the breast were stained using immunohistochemistry for von Willebrand factor, CD3, CD8, CD45RO, CD45RA, CD20, CD68, and c-erbB-2. Tumour vascularity was assessed by counting vessels in the three most vascular areas, and calculating the average (x 400 magnification, field 0.168 mm2). Each pattern of inflammation was scored semiquantitatively. RESULTS: The main pattern of inflammation was a diffuse infiltrate of macrophages, and to a lesser extent T cells. Perivascular and perilobular clusters of B and T cells were noted at the edge of the carcinomas, but were less prominent than the diffuse inflammation. Diffuse inflammation, particularly macrophages, was associated with high tumour grade, tumour necrosis, large tumour size, and c-erbB-2 expression. Perivascular and perilobular inflammation also increased with tumour grade. Tumour vascularity increased slightly with intensity of diffuse inflammation (Spearman's rank correlation coefficient rs = 0.17, p = 0.08), and was inversely related to perilobular inflammation (rs = -0.23, p = 0.03). CONCLUSIONS: The correlations between inflammation and vascularity were weak in this study (r2 about 0.04) and thus there was no evidence of an important relation. Discrepancies between this and other studies may be resolved by studying expression of angiogenic cytokines and proteolytic enzymes by tumour infiltrating inflammatory cells, and their relation to tumour vascularity.

Angiogenesis and inflammation in ductal carcinoma in situ of the breast.

Several recent studies suggest that vascular density may be an independent prognostic indicator in invasive carcinoma of the breast. Increased vascularity has also been shown in ductal carcinoma in situ (DCIS). The prognostic significance of the inflammatory infiltrate in mammary carcinoma is more controversial, but it could affect angiogenesis by releasing angiogenic factors and digesting matrix. Vascularity and inflammation have been studied in 41 examples of pure DCIS, classified using the method of Holland et al. Immunohistochemistry was performed with antibodies to von Willebrand factor, CD3, CD8, CD45RO, CD45RA, CD20, CD68, and c-erB-2. The main pattern of inflammation was clusters of B and T cells situated either adjacent to involved ducts or in the interductal stroma. Typically, these clusters were around vessels with plump endothelium suggestive of high endothelial venules. A less prominent pattern was a diffuse stromal infiltrate of macrophages and T cells. There were two patterns of increased vascularity associated with DCIS: necklaces of vessels close to the involved ducts and vessels arranged diffusely in the interductal stroma. Each pattern of inflammation and of vascularity was graded semi-quantitatively. Increased stromal vascularity was associated with the perivascular clusters of inflammation; both were associated with c-erB-2 expression and extent of the DCIS. Necklaces of vessels were associated with the diffuse inflammation. Perivascular inflammation and c-erB-2 (but neither pattern of vascularity) were associated with poor differentiation of the DCIS. Thus, different patterns of inflammation are associated with different patterns of vessels. The clusters of B and T cells may be recruited via high endothelial venules induced by the DCIS. Cytokines released by the DCIS and/or the inflammatory cells (clusters or diffuse) may stimulate the two patterns of new vessel formation.

c-erbB-3 protein expression in ductal carcinoma in situ of the breast.

c-erbB-3, A recently identified member of the type I tyrosine kinase receptor family, has been shown to be overexpressed in invasive ductal carcinoma of breast. In this study, expression of the c-erbB-3 protein was examined in 57 cases of pure ductal carcinoma in situ of the breast (DCIS) by immuno-cytochemical methods. Staining was either absent (17 cases), present at levels equivalent to that found in adjacent normal tissue (20) or greater than in normal tissue (20). In most cases the pattern of staining was cytoplasmic, but in 4 cases with the most intense reaction there was also focal membrane staining. In the same series of cases, c-erbB-2 protein had previously been shown to be overexpressed in 28 of 57 cases, c-erbB-2 overexpression was correlated with normal level of c-erbB-3, and lack of c-erbB-2 expression was correlated with c-erbB-3 overexpression.

Inflammatory infiltrate in invasive lobular and ductal carcinoma of the breast.

The significance of inflammation in carcinoma of the breast is controversial. Little attention has been paid to different patterns of inflammation or inflammation associated with different histological types of carcinoma. We have looked at the pattern of inflammation in 123 invasive mammary carcinomas (including 46 lobular), and characterised the inflammatory cells with immunohistochemistry in 21. We found different patterns of inflammation in ductal and lobular carcinoma. Diffuse inflammation was seen more in ductal carcinoma, particularly of high grade, and was predominantly composed of macrophages and T cells. It was associated with necrosis, but the correlation was weak, suggesting that other factors are important. Perilobular inflammation was seen most frequently in lobular and high-grade ductal carcinomas, particularly at the tumour edge. Perivascular inflammation was also largely at the tumour edge, but was not more common in any tumour type. In contrast to the diffuse inflammation, the perivascular and perilobular inflammation was composed of T and B cells. Normal lobules at the tumour edge showed consistent expression of HLA-DR, whereas lobules away from the tumour were negative. A combination of perilobular and perivascular inflammation composed of B and T cells with epithelial expression of HLA-DR mimicking lymphocytic lobulitis was seen more frequently in lobular than ductal carcinoma.

Adhesion molecule expression and leucocyte trafficking following immunotherapy with recombinant interleukin-2.

The relevant anti-tumour mechanisms of recombinant interleukin-2 (rIL-2) in vivo are unclear but an influx of T-lymphocytes and macrophages has been noted in regressing lesions. One of the dose limiting toxicities of rIL-2 is the development of a capillary leak syndrome attributed to widespread endothelial activation. Changes in expression of endothelial and leucocyte-associated adhesion molecules were assessed in tumour and uninvolved skin in patients with metastatic breast cancer receiving rIL-2. Increased expression of intracellular adhesion molecule-1, its leucocyte-associated ligand, leucocyte function associated molecule-1, vascular cell adhesion molecule and its ligand, very late after activation antigen-4 as well as members of the selectin family of adhesion molecules, were noted in uninvolved skin following rIL-2. Expression of these adhesion molecules was noted in tumour stroma before rIL-2 but little change was observed following rIL-2 infusion. An influx of monocytes and T-lymphocytes (expressing the IL-2 receptor and of the memory subtype) and a lower number of neutrophils was noted in uninvolved skin following rIL-2. Although monocytes and T-lymphocytes were present in tumour stroma before rIL-2 no changes were observed following infusion. The changes noted in the dermis contrast with those seen at tumour sites and may partly explain the low therapeutic index of rIL-2.

Tissue plasminogen activator production by monocytes in venous thrombolysis.

Laminated occlusive thrombus was induced in the rat inferior vena cava (IVC) by a distal stenosis and injection of thrombin. Immunocytochemistry was performed on serial cryostat sections of the thrombus for tissue plasminogen activator (tPA) and a variety of phenotype markers for mononuclear cells. There was little tPA in 2-day-old thrombus. However, tPA was present in significant quantities in 1- and 2-week-old thrombus. Most of the staining for tPA was associated with monocytes, which had infiltrated the thrombus in large numbers. No caval endothelium was seen in these sections. By 4 weeks, the IVC had re-canalized and new endothelium had formed; tPA staining was weakly positive in the endothelium and smooth muscle. In situ hybridization with a digoxigenin-labelled RNA probe confirmed the monocytes as the main source of tPA. This study shows that large numbers of infiltrating monocytes are present in venous thrombosis and that they are the main source of tPA.

The tissue plasminogen activator and urokinase response in vivo during natural resolution of venous thrombus.

PURPOSE: The aim of this study was to measure the distribution of endogenous plasminogen activators during thrombolysis with an endothelial-conserving model of laminated thrombosis. METHODS: Thrombi were raised in the inferior vena cava of rats with thrombin and flow reduction. The thrombi, adjacent vein wall, and distant veins (the superior vena cava) were removed at intervals from 1 hour to 21 days from formation and then cryohomogenized and assayed with specific bioimmunoassays for tissue-type (t-PA) and urokinase-type plasminogen activators (u-PA). RESULTS: The measured t-PA activity of the vein wall around the thrombus was reduced compared with the control inferior vena cava at 4 days. Both the u-PA and t-PA content of the thrombus increased progressively during thrombolysis. The t-PA activity increased significantly in the distant vein walls in the animals with thrombi. Immunocytochemistry and in situ hybridization localized the t-PA to a mononuclear cell infiltrate and showed up-regulation of mRNA for rat t-PA in these monocytes. CONCLUSIONS: The local plasminogen activator response was predominantly within the thrombus itself. Increased t-PA activity was additionally found in distant veins but was reduced in the vessel wall adjacent to the thrombus. This is the first report to show that u-PA activity is increased within organizing thrombus in vivo and that most of the t-PA activity is localized to a monocyte infiltrate.